The ETS-related gene (ERG) proto-oncogene is overexpressed in 60-70% of prostate tumors as a result of recurring gene fusions involving TMPRSS2 and the ETS family of genes (Petrovics et al., 2005; Tomlins et al., 2005; reviewed in Kumar-Sinha et al., 2008). Emerging studies on human prostate cancer specimens and various experimental models underscore the causative oncogenic function of ERG in prostate cancer (Klezovitch et al., 2008; Tomlins et al., 2008; Sun et al., 2008; Wang et al., 2008). Numerous reports highlighted both diagnostic and prognostic features of the genomic activation of ERG revealing that about half the prostate tumors harbor the most common gene fusion that takes place between the androgen receptor-regulated TMPRSS2 gene promoter and ERG protein coding sequence (reviewed in Kumar-Sinha et al., 2008). Fusion between the TMPRSS2 gene promoter and ERG results in the overexpression of N-terminally truncated or full-length forms of ERG (Klezovitch et al., 2008; Sun et al., 2008). Fusion events between erg and other androgen inducible promoter sequences, such as SLC45A3 (Han et al., 2008) and NDRG1 (Pflueger et al., 2009), have also been identified in prostate cancer.
Poor disease outcome for patients with tumors harboring duplications of TMPRSS2/ERG fusions or chromosomal losses (Edel) associated with the fusion event has been highlighted (Attard et al., 2008; FitzGerald et al., 2008; Mehra et al., 2008). Current diagnosis of prostate cancer is based on a variety of histological features, including architectural growth pattern, loss of basal cells, nuclear atypia, amphophilic cytoplasm, intraluminal blue mucin, pink amorphous secretion, and mitotic figures (Egevad, 2008). If some of these features are not apparent, it can be difficult for pathologists to diagnose prostate cancer, especially in the case of prostate needle biopsies with very limited tumor content (Mostofi et al., 1992; Mostofi et al., 1993). Molecular markers are now being used to aid diagnosis. For example, prostate cancer diagnosis can include basal cell staining of benign glands for specific cytokeratin or p63 (CK903, p63) and tumor cell associated alpha-methyl acyl-CoA-Racemase (AMACR or P504S) (Luo et al., 2002; Rubin et al., 2002). However, these molecular markers have marked limitations in routine diagnosis. Expression of AMACR is found in a variety of other non-malignant lesions, including up to 21% of benign prostatic glands, 58% of nephrogenic adenomas and approximately 18% of cases of atypical adenomatous hyperplasia (Beach et al., 2002; Gupta et al., 2004; Jiang et al., 2001; Yang et al., 2002). New data evaluating ERG over expression and TMPRSS2/ERG genomic rearrangement are providing highly promising new strategies in prostate cancer diagnosis and prognosis (Furusato et al., 2008; Saramaki et al., 2008).
EWS-ERG fusions have been described in a small subset of Ewing's sarcoma, whereas ERG overexpression without fusion was highlighted in acute myeloid leukemia and acute T-lymphoblastic leukemia (Marcucci et al., 2005; Baldus et al., 2006). ERG overexpression has also been linked to megakaryoblastic leukemia (Rainis et el. 2005). Other studies suggest that increased ERG expression plays a role in Alzheimer's Disease (AD) and AD-like neuropathy in Down Syndrome (Shim et al., 2003; Ng et al. 2010).
The structure of the human ERG gene includes at least 17 exons spanning approximately 300 kilobases of genomic sequence and generating at least nine separate transcripts. (Owczarek et al., 2004). The ERG1-ERG5 isoforms encode five separate polypeptides that can bind the ETS site and act as transcriptional activators. (Owczarek et al., 2004). Of these five isoforms, ERG3 is the longest, encoding a 479 amino acid polypeptide (SEQ ID NO:1; Accession No. NP—891548.1). The ERG6-ERG9 isoforms represent alternative splice forms with a different 5′ exon from other ERG isoforms. While ERG7 and ERG8 have open reading frames, ERG6 encodes multiple stop codons, suggesting that this ERG transcript does not code for a functional protein. (Owczarek et al., 2004). The ERG9 transcript does not contain a putative start codon or a consensus polyadenylation signal suggesting that it might also be a non-coding transcript. (Owczarek et al., 2004).
Although, the ERG proto-oncogene was initially characterized more than twenty years ago (Rao et al., 1987a; Rao et al., 1987b; Reddy et al., 1987), currently there is no available antibody for detecting ERG in clinical specimens. The ERG protein belongs to a highly homologous group of proteins, the ETS (E-twenty six specific, E26 transformation specific) multi-gene family of transcription factors, which are conserved throughout the metazoans (Turner and Watson, 2008). ETS proteins contain a winged helix-turn-helix DNA binding domain and a pointed (SAM) domain implicated in protein-protein interaction. The high degree of homology between members of this family presents a significant obstacle for raising an antibody against a specific member of this protein family.
Although polyclonal ERG antibodies are commercially available, these antibodies exhibit low affinity for endogenous levels of the ERG protein and high levels of non-specific staining that limit their usefulness, for example, in immunohistochemistry testing. As such, the commercially available polyclonal ERG antibodies are not suitable for detecting ERG proteins in clinical settings (e.g., in a tissue biopsy).